Nucleic acid sequence analysis is becoming increasingly important in many research, medical, and industrial fields, e.g. Caskey, Science 236:1223-1228 (1987); Landegren et al, Science, 242:229-237 (1988); and Arnheim et al, Ann. Rev. Biochem., 61:131-156 (1992). In particular, ligation-based techniques, such as oligonucleotide ligation assay (OLA), ligation chain reaction (LCR), ligation amplification reaction (LAR), and the like, form an important family of sequence analysis tools, e.g. Barany, PCR Methods and Applications 1:5-16 (1991); Landegren et al, U.S. Pat. No. 4,988,617; Landegren et al, Science 241: 1077-1080 (1988); Backman et al, European patent publication 0439182A2; Whiteley et al, U.S. Pat. No. 4,883,750; Yu and Wallace, Genomics 4:560-569 (1989); Nickerson et al, Proc. Natl. Acad. Sci. 87:8923-8927 (1990); and the like.
Ligation-based techniques rely on oligonucleotides annealing to contiguous regions of a target sequence. If there is perfect complementarity between the target sequence and the oligonucleotides at the junction between the oligonucleotides, then ligation can be effected. If the terminal nucleotide of either oligonucleotide is not complementary with its corresponding nucleotide on the target sequence at the junction, then ligation cannot be effected. A key feature of ligation-based assays is the ability of the ligation reaction, whether chemical or enzymatic in nature, to distinguish between mispairing at the junction and perfect complementarity at the junction. However, even if the terminal nucleotides are complementary with their corresponding target nucleotides, ligation can still fail if one of the terminal nucleotides has a substantial residence time in a non-annealed equilibrium state, for example, as may occur with AT-rich termini or ligation is attempted at high temperature. The result is a loss of sensitivity in the assay employing the ligation.
The sensitivity of ligation-based techniques for analyzing nucleotide sequences would be substantially increased with the availability of a method for enhancing the stability of oligonucleotides hybridized to a complementary target sequence.